We have previously described a C-terminally truncated form of CPSF6 (CPSF6-358) that has potent antiviral activity against different HIV-1, HIV-2, and SIV isolates, but no activity against EIAV, FIV, or MLV. CPSF6-358 interferes with the nuclear entry of HIV-1. We selected for HIV-1 replication in cells expressing CPSF6-358 and identified a mutation of N74D of CA as sufficient to confer viral resistance. We examined the nuclear pore dependencies for wild-type (WT) and N74D HIV-1 and found that the isolates require some of the same pore proteins but differ in requirements for others. In particular, N74D HIV-1 was less sensitive to depletion of TNPO3 (a karyopherin), NUP153, and NUP358/RanBP2 than WT HIV-1. These experiments established a genetic interaction between HIV-1 CA and the nuclear pore. We are now attempting to define the route that HIV-1 takes to the nucleus: the cell factors it interacts with and how it interacts with these factors. Our data suggest a direct interaction of WT CPSF6 with CA. We have also discovered that TNPO3 regulates CPSF6 nuclear localization. We are examining whether CPSF6 normally interacts with HIV-1 CA in the cytoplasm and whether this interaction is important for nuclear entry of the virus. Within the nucleus, we find that the HIV-1 pre-integration complex interacts with CPSF6, and this interaction regulates HIV-1 integration in the host cell chromatin. Notably, we find that by interacting with CPSF6, HIV-1 is first directed to speckles after entering the nucleus and prior to integration. Nuclear speckles colocalize with regions of chromatin that HIV-1 preferentially integrates. Thus, we hypothesize that HIV-1 co-opted CPSF6 to access regions of chromatin that are broadly transcriptionally active due to their proximity to nuclear speckles.